This proposal outlines biochemical and genetic experiments that will distinguish replicative functions of the multiple DNA-dependent DNA polymerases extracted from cultured mammalian cells. The approach involves generating in culture temperature-sensitive replication mutants, and examining their content of DNA polymerases in vitro. The chromatographic, enzymatic and immunologic properties of DNA polymerases purified from somatic cell mutants will be compared directly to analogous polymerases of the wild-type parental cell clone. The biochemical and physical properties of each DNA polymerase, in conjunction with the growth characteristics of appropriate mutants, should indicate functions for each polymerase in both replicative and non-replicative DNA metabolism. A model, biochemically defined, replicative reaction will be purified from animal cells infected with a cytoplasmic DNA virus, FV-3. This virus contains a linear duplex DNA-genome of documented physical and chemical properties, grows to high titers in many mammalian cells in culture and, most importantly, enhances dramatically the level of DNA polymerase extracted from the cell's cytoplasm. Enzymes purified from FV-3-infected cells will be used to "replicate" viral DNA in vitro. The purified viral DNA replicating reaction will provide in vitro a model assay for macromolecular components isolated from replication-defective cell mutants, from wild-type cells and from cells infected with wild-type and mutant Frog Virus 3.